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Am J Physiol Cell Physiol 294: C754-C764, 2008. First published January 16, 2008; doi:10.1152/ajpcell.00494.2007
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RECEPTORS AND SIGNAL TRANSDUCTION

Trauma-hemorrhage inhibits splenic dendritic cell proinflammatory cytokine production via a mitogen-activated protein kinase process

Takashi Kawasaki,1 Mashkoor A. Choudhry,1 Martin G. Schwacha,1 Satoshi Fujimi,2 James A. Lederer,2 Kirby I. Bland,1 and Irshad H. Chaudry1

1Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama; and 2Department of Surgery (Immunology), Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 18 October 2007 ; accepted in final form 13 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although splenic dendritic cell (DC) functions are markedly altered following trauma-hemorrhage, the mechanism(s) responsible for the altered DC functions remains unknown. We hypothesized that trauma-hemorrhage inhibits DC function via suppressing toll-like receptor 4 (TLR4) expression and mitogen-activated protein kinases (MAPKs). To examine this, male C3H/HeN (6–8 wk) mice were randomly assigned to sham operation or trauma-hemorrhage. Trauma-hemorrhage was induced by midline laparotomy and ~90 min of hypotension [blood pressure (BP) 35 mmHg], followed by fluid resuscitation (4x the shed blood volume in the form of Ringer lactate). Two hours later, mice were euthanized, splenic DCs were isolated, and the changes in their MAPK activation, TLR4-MD-2 expression, and ability to produce cytokines were measured. The results indicate that trauma-hemorrhage downregulated the lipopolysaccharide (LPS)-induced MAPK activation in splenic DCs. In addition to the decrease in MAPK activation, surface expression of TLR4-MD-2 was suppressed following trauma-hemorrhage. Furthermore, LPS-induced cytokine production from splenic DCs was also suppressed following trauma-hemorrhage. These findings thus suggest that the decrease in TLR4-MD-2 and MAPK activation may contribute to the LPS hyporesponsiveness of splenic DCs following trauma-hemorrhage. Hyporesponsiveness of splenic DCs was also found after stimulation with the TLR2 agonist zymosan. Our results may thus explain the profound immunosuppression that is known to occur under those conditions.

toll-like receptor 4 expression; splenic dendritic cell; toll-like receptors; lipopolysaccharide

STUDIES INDICATE THAT TRAUMATIC INJURY induces immune dysfunction that is associated with an increased susceptibility to sepsis, organ failure, and mortality (8, 29, 35, 41, 50). Such conditions remain the leading causes of death after trauma despite the improvement in medical management over the past decades. Increased susceptibility to infection and higher risk of complications are secondary to the inhibitory effect of trauma-hemorrhage on cell-mediated immune responses and microbicidal activity by cells of the innate immune system (38, 45). It is well known that trauma-hemorrhage induces a marked alteration of many immune functions, including T-cell activation, proliferation, and cytokine release, and macrophage antigen presentation function and cytokine release (6).

DCs are potent antigen-presenting cells that can stimulate T-cells in the primary immune response (46). Immature DCs are located in almost all tissues, where they can capture and process antigens. DC maturation is associated with enhanced production of inflammatory cytokines and chemokines, reduced ability to internalize antigens and with acquisition of migratory functions that allow antigen-loaded DCs to migrate toward the T-cell areas of secondary lymphoid organs (17). Mature DCs show high surface expression of both costimulatory and adhesion molecules, including CD40, CD80, and CD86, as well as major histocompatibility complex class II antigens (7). CD83, a specific marker for DC maturation, is also upregulated in mature DCs (9). The mature DCs also have the ability to activate both T helper 1 (Th1) and Th2 cell responses. Our previous studies have shown that splenic DC functions are also suppressed following trauma-hemorrhage (28).

Lipopolysaccharide (LPS) is a well-known and potent activator of DCs, and it induces the production of proinflammatory cytokines such as IL-1, tumor necrosis factor (TNF)-{alpha}, and IL-12. Innate immune responses mediated by toll-like receptors (TLRs) are the first line of defense against infectious agents entering the organism (37). TLR4 has been shown to be essential for cellular responsiveness to LPS (22, 43). Previous studies have shown that activation of mitogen-activated protein kinases (MAPKs) is important for the regulation of maturation, survival, and cytokine secretion by DCs (1, 40, 52). There are three major families of MAPKs: p38 MAPK, extracellular signal-regulated protein kinase 1 (ERK1) and ERK2, and c-Jun NH2-terminal kinase/stress-activated protein kinases (JNK/SAPK). LPS activates all three families of MAPKs. After LPS stimulation, the phosphorylated MAPKs transduce their signals downstream and promote activation and translocation of transcription factors that subsequently regulate the expression of different genes and biological functions of DCs. Although our previous study (28) has shown that LPS-induced cytokine production is suppressed following trauma-hemorrhage, it remains unknown whether trauma-hemorrhage has any effect on TLR expression and MAPK activation of splenic DCs. We hypothesized that trauma-hemorrhage decreases splenic DC TLR expression and MAPK activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. Male C3H/HeN mice (Charles River Laboratories, Wilmington, MA) 6–8 wk, weighing 20–25 g, were used in the experiments. The mice were allowed to acclimatize in our animal facility for 1 wk before experimentation. All animal experiments were conducted in accordance with guidelines set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH) and approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.

Trauma-hemorrhage. Mice were fasted overnight but were allowed water ad libitum. They were anesthetized with isoflurane (Attane; Minrad, Buffalo, NY) and restrained in supine position. A 2.0-cm midline laparotomy (i.e., induction of soft tissue trauma) was performed, and the abdominal incision was then closed aseptically in two layers using 6-0 Ethilon sutures (Ethicon, Somerville, NJ). Both femoral arteries were then aseptically catheterized with polyethylene-10 tubing (Clay-Adams, Parsippany, NJ), and the animals were allowed to awaken. Blood pressure was monitored continuously through one of the femoral catheters using a blood pressure analyzer (Digi-Med BPA-190; Micro-Med, Louisville, KY). Upon awakening, the animals were bled rapidly through the other catheter to a mean arterial pressure of 35 ± 5 mmHg that was maintained for 90 min. At the end of that period, animals were resuscitated with four times the shed blood volume in the form of lactated Ringer solution over 30 min. Blood pressure was monitored continuously until it recovered to normal level (over 80 mmHg). Lidocaine was applied to the groin incision sites, the catheters were removed, the vessels were ligated, and the incisions were closed. The mice were then returned to the cage. Sham-treated animals underwent the same anesthetic and surgical procedures, but neither hemorrhage nor fluid resuscitation was performed. The animals were anesthetized by isoflurane administration 2 h after trauma-hemorrhage, at which time blood and spleen were collected for analysis.

Isolation of splenic dendritic cells. Spleens were digested by Liberase CI (Roche, Indianapolis, IN) and teased apart by repeated pipetting in PBS containing 5% FCS and 5 mM EDTA. The red blood cells were osmotically lysed, and splenocytes were blocked with 1 µg/ml Fc block (clone: 93) antibody for 15 min on ice. Cell suspensions were enriched with anti-CD11c magnetic beads and positive selection columns MS+ according to manufacturer's instructions (Miltenyi Biotec, Auburn, CA). Flow cytometric analysis demonstrated that cells contained >90% CD11c-positive cells.

Determination of cytokines level in CD11c-positive cell culture supernatants. Purified CD11c-positive cells (1 x 105 cells/well) from the spleen were cultured in 96-well tissue culture plates in RPMI-1640 medium containing 10% heat-inactivated FBS and antibiotics, 100 U/ml penicillin, and 100 µg/ml streptomycin, with or without the TLR4 ligand LPS from Escherichia coli 0111:B4 (10 µg/ml, Sigma-Aldrich, St. Louis, MO). In some experiments, cells were stimulated with the TLR2 ligand zymosan from Saccharomyses cerevisiae (10 µg/ml, InvivoGen, San Diego, CA). After 24 h incubation at 37°C, 5% CO2, the plate was centrifuged at 400 g for 10 min. The dose and time course of LPS stimulation was the same as used in our previous study (28). Supernatants were collected and frozen at –80°C until analysis. The concentrations of IL-6, IL-10, IL-12p70, and TNF-{alpha} in DC supernatant were measured using a Cytometric Bead Array (CBA) mouse inflammation kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. Briefly, 50 µl of mixed capture beads were incubated with 50 µl of supernatant and 50 µl phycoerythrin (PE) detection reagent for 2 h at room temperature. The immunocomplexes were then washed and analyzed using the LSRII flow cytometer (BD Biosciences, Mountain View, CA). Data processing was carried out using the accompanying fluorescein-activated cell sorter (FACS) Diva (FACS Diva) and BD CBA software. IL-12p40 concentrations in cell supernatant were measured by ELISA (BD Biosciences).

Measurement of total MAPK signaling molecules by fluorescein-activated cell sorter analysis. At 2 h after resuscitation, splenic CD11c-positive cells were isolated from mice as described above. The cells were then incubated with 10 µg/ml LPS for 0, 5, 15, 30, or 60 min. The cells were fixed with 1.5% paraformaldehyde (PAF) for 10 min at 37°C, permeabilized with ice-cold methanol (100%) for an additional 10 min, and washed with phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin and 0.1% sodium azide (PBA). The fixed cells were stained with unlabeled primary rabbit polyclonal antibodies specific for p38, extracellular signal-related protein kinase (ERK), or stress-activated protein kinase (SAPK/c-Jun) NH2-terminal kinase (JNK) (SAPK/JNK) (Cell Signaling, Beverly, MA) as previously described (33). An isotype-matched rabbit IgG was used as a nonspecific staining control. The cells were then washed and stained with a goat anti-rabbit PE-labeled secondary antibody (Jackson ImmunoResearch, West Grove, PA) and subsequently washed twice and resuspended in 0.3% PAF. Flow cytometry was performed using the LSRII instrument (BD Biosciences), and the results were analyzed using the accompanying FACS Diva software.

Detection of phosphorylated (activated) MAPK signaling molecules by FACS. To distinguish between the activated and nonactivated forms of MAPK, we used antibodies specific for the phosphorylated (active) forms of p38, ERK1/2, and SAPK/JNK (Cell Signaling) as previously described (33). At 2 h after resuscitation, splenic CD11c-positive cells were isolated and were then incubated with 10 µg/ml LPS. After 0-, 5-, 15-, 30-, or 60-min incubation periods, the cells were rapidly fixed with 1.5% PAF, permeabilized with methanol, and stained and analyzed as described above using antibodies specific for the phosphorylated MAPKs. An isotype-matched rabbit IgG was used as a nonspecific staining control. The cells were then washed and stained with a goat anti-rabbit PE-labeled secondary antibody (Jackson ImmunoResearch). This was followed by two additional washings of the cells; they were then resuspended in 0.3% PAF. Flow cytometry was performed using the LSRII instrument (BD Biosciences), and the results were analyzed using the accompanying FACSDiva software.

Measurement of cytokine production following MAPK inhibition. Splenic CD11c-positive cells were isolated and treated with MAPK inhibitors. Briefly, the CD11c-positive cells were treated with selective inhibitors: a highly specific and cell-permeable inhibitor of p38 MAPK (SB203580; 5 µM; IC50 = 600 nM); a selective and cell-permeable inhibitor of MAPK kinase (MAPKK) that acts by inhibiting the activation of ERK1/2 and subsequent phosphorylation of MAPK (PD98059; 20 µM; IC50 = 2 µM); and a potent, cell-permeable, selective and reversible inhibitor of JNK (JNK Inhibitor II/SP600125; 20 µM; IC50 = 40 nM for JNK-1 and JNK-2 and 90 nM for JNK-3) (all from Calbiochem, La Jolla, CA) for 15 min before stimulation as previously described (5, 48). The cells were then cultured with LPS (10 µg/ml) for 24 h at 37°C, 5% CO2, cell-free culture supernatants harvested and frozen at –80°C until further analysis. In some experiments, cells were stimulated with zymosan (10 µg/ml) for 24 h.

FACS analysis of cell-surface TLR4-MD-2 expression. Cell-surface TLR4-MD-2 levels were measured using flow cytometry. Splenic CD11c-positive cells from mice were isolated as described above. The cells were then stained with PE-labeled anti-TLR4-MD-2 antibodies (eBiosciences). The cells were washed twice with PBA, stored in 0.3% PAF, and subsequently analyzed by flow cytometry.

Extraction of total RNA and cDNA synthesis. Total RNA was extracted from isolated splenic DCs using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Aliquots of 0.5 µg of total RNA were reversed to cDNA using a commercially available reverse-transcriptional kit (TaqMan Reverse Transcription Reagents; Applied Biosystems, Foster City, CA) in a 50-µl reaction mix.

Quantitative real-time PCR. The expression levels of TLR4 and β2-microglobulin mRNAs were determined by real-time PCR using cells from experimental and sham animals that were harvested but not stimulated with LPS in vitro. This was carried out in a 20-ml reaction mix containing 9 ml of cDNA, 10 ml of 2 X TaqMan Universal PCR Master Mix (Applied Biosystems), and 1 ml of primers and TaqMan probe mix (Applied Biosystems). The following validated primers and TaqMan MGB probes (6-FAM labeled) were used: TLR4 (assay ID: Mm00445274_m1) and β2-microglobulin (Mm00437762_m1). All real-time PCR assays were performed in duplicate using an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) with the following program: 50°C for 2 min to activate uracil N-glycosylase enzyme, 95°C for 10 min to denature uracil N-glycosylase and activate DNA polymerase, 45 cycles at 95°C for 20 s, and at 60°C for 1 min. β2-Microglobulin was used as an internal control. The relative expression levels of individual target genes were normalized to the internal control.

Statistical analysis. The data were presented as means ± SE. One-way analysis of variance and Tukey's test were used for the comparison between groups, and the differences were considered to be significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of trauma-hemorrhage on LPS-induced proinflammatory cytokines production by splenic DCs. Splenic DCs from trauma-hemorrhage mice produced lower levels of tumor necrosis factor-{alpha} (TNF-{alpha}) in response to LPS stimulation compared with cells from sham mice (P < 0.05; Fig. 1A). LPS-stimulated IL-6 production was also suppressed in splenic DCs from trauma-hemorrhage mice (P < 0.05; Fig. 1B) compared with cells from sham animals. In addition, LPS-stimulated IL-12p40 production was suppressed following trauma-hemorrhage (P < 0.05; Fig. 1D). In contrast to TNF-{alpha}, IL-6, and IL-12p40, trauma-hemorrhage had no effect on LPS-stimulated IL-10 production by splenic DCs (Fig. 1C). Furthermore, no difference was observed in TNF-{alpha}, IL-6, IL-10, and IL-12 in the supernatants harvested from unstimulated DCs from trauma-hemorrhage and sham mice. These results are consistent with our previous study (28) that also demonstrated trauma-hemorrhage induced LPS-hyporesponsiveness of splenic DCs.


Figure 1
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  		Fig. 1. Tumor necrosis factor (TNF)-{alpha}, IL-6, IL-10, and IL-12 production by splenic dendritic cells (DCs) following trauma-hemorrhage. Two hours after resuscitation, DCs were purified from splenocytes, and the purified CD11c-positive cells were cultured in 96-well tissue culture plates with LPS (10 µg/ml) for 24 h. The levels of TNF-{alpha} (A), IL-6 (B), IL-10 (C), and IL-12p70 (D) in DC supernatant were measured using Cytometric Bead Array (CBA). IL-12p40 (D) levels were measured by ELISA. Data are shown as means ± SE of 6 animals in each group. *P < 0.05 compared with the equivalent sham group; #P < 0.05 compared with nonstimulation group (–).

 
Effect of trauma-hemorrhage on activation of p38, ERK1/2, and SAPK/JNK. To evaluate activation of p38, ERK1/2, and SAPK/JNK, we used a flow cytometric approach as previously described (33). Phosphorylation of p38, ERK1/2, and SAPK/JNK was detected in isolated splenic DCs in the absence of LPS stimulation. Unstimulated cells did not exhibit any significant differences in the levels of phosphorylated p38, ERK1/2, and SAPK/JNK in DC from sham and trauma-hemorrhage mice (data not shown).

Effect of trauma-hemorrhage on LPS-stimulated p38, ERK1/2, and SAPK/JNK activation. Since LPS was used to stimulate cytokine production by splenic DCs, we examined whether trauma-hemorrhage-induced inhibition of cytokine production resulted from the impaired phosphorylation of MAPK in response to LPS. Thus we compared LPS-induced activation of MAPK in splenic DCs isolated from mice 2 h after resuscitation. LPS stimulation significantly increased (P < 0.05) the expression of the active (phosphorylated) form of p38 MAPK in cells from both sham and trauma-hemorrhage mice (Fig. 2A). Maximal activation of p38 MAPK in trauma-hemorrhage DCs was observed at 15 min after LPS stimulation. In DCs from sham mice a similar pattern was observed; however, the activation state persisted even at 30 min after LPS stimulation. In both cell populations, activated p38 MAPK returned to basal levels at 60 min after LPS stimulation. Total p38 MAPK expression was not significantly different between DCs from sham and trauma-hemorrhage mice at any time points after LPS stimulation (Fig. 2B). The activated p38 MAPK-positive cell percentage at 15 min after LPS stimulation was significantly decreased in the trauma-hemorrhage group (Fig. 2D).


Figure 2
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  		Fig. 2. LPS-stimulated splenic DC p38 mitogen-activated protein kinase (MAPK) activation. Splenic DCs were isolated 2 h after resuscitation or sham procedure and stimulated with LPS (10 µg/ml) for 0–60 min. Phosphorylated p38 MAPK (A) and total p38 MAPK (B) were determined by flow cytometry as described in MATERIALS AND METHODS. A representative fluorescein-activated cell sorter (FACS) result of phosphorylated p38 MAPK with or without LPS stimulation for 15 min is shown in (C). Percentage of phosphorylated p38 MAPK-positive cells after stimulation with or without LPS for 15 min are also shown in (D). MFI, mean fluorescence intensity. Data are means ± SE for 6 mice/group per time point. #P < 0.05 compared with no stimulation (i.e., 0 min); *P < 0.05 compared with respective sham group.

 
LPS stimulation also significantly increased (P < 0.05) the expression of the active form of ERK1/2 in cells from both sham and trauma-hemorrhage mice (Fig. 3A). Maximal activation of ERK1/2 was observed at 15 min after LPS stimulation. In DCs from sham mice, the level of phosphorylated ERK1/2 was significantly greater (P < 0.05) than that from trauma-hemorrhage DCs. Total ERK1/2 expression was not significantly different between DCs from sham or trauma-hemorrhage mice; however, there was a trend toward higher expression in cells from sham mice that did not reach statistical significance (Fig. 3B). The percentage of phosphorylated ERK1/2-positive cells revealed the decrease in ERK1/2 activation following trauma-hemorrhage (Fig. 3D).


Figure 3
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  		Fig. 3. LPS-stimulated splenic DC extracellular signal-regulated protein kinase (ERK) 1/2 activation. Splenic DCs were isolated 2 h after resuscitation or sham procedure and stimulated with LPS (10 µg/ml) for 0–60 min. Phosphorylated ERK1/2 (A) and total ERK1/2 (B) were determined by flow cytometry as described in MATERIALS AND METHODS. C: representative FACS result of phosphorylated ERK1/2 with or without LPS stimulation for 15 min. D: percentage of phosphorylated ERK1/2-positive cells after stimulation with or without LPS for 15 min. Data are means ± SE for 6 mice/group per time point. #P < 0.05 compared with no stimulation (i.e., 0 min); *P < 0.05 compared with respective sham group.

 
Although there was a trend toward lower expression of phosphorylated SAPK/JNK in splenic DCs from trauma-hemorrhage mice after LPS stimulation, the values were not significantly different (Fig. 4A). A trend toward lower expression of total SAPK/JNK levels was also observed in DCs from trauma-hemorrhage mice; however, the values were not significant in DCs from sham or trauma-hemorrhage mice at any time point after LPS stimulation (Fig. 4B). The percentage of phosphorylated APK/JNK cells increased after LPS stimulation; however, there was no significant difference between the two groups (Fig. 4D).


Figure 4
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  		Fig. 4. LPS-stimulated splenic DC stress-activated protein kinases (SAPK)/c-Jun NH2-terminal kinase (JNK) (SAPK/JNK) activation. Splenic DCs were isolated 2 h after resuscitation or sham procedure and stimulated with LPS (10 µg/ml) for 0–60 min. Phosphorylated SAPK/JNK (A) and total SAPK/JNK (B) were determined by flow cytometry as described in MATERIALS AND METHODS. C: representative FACS result of phosphorylated SAPK/JNK with or without LPS stimulation for 15 min. D: percentage of phosphorylated SAPK/JNK-positive cells after stimulation with or without LPS for 15 min. Data are means ± SE for 6 mice/group per time point. #P < 0.05 compared with no stimulation (i.e., 0 min); *P < 0.05 compared with respective sham group.

 
Effect of p38 MAPK, ERK1/2, and SAPK/JNK inhibition on splenic DC LPS-induced cytokines production. Our results showed that MAPK activation is suppressed following trauma-hemorrhage; however, MAPK activation increased after LPS stimulation compared with no stimulation even in the splenic DCs from trauma-hemorrhage mice. These results thus indicate that MAPK activation is not completely suppressed in our trauma-hemorrhage model. Therefore, to further ascertain the role of MAPKs in the suppression of TNF-{alpha}, IL-6, and IL-12p40 production in trauma-hemorrhage mice, we examined whether inhibition of p38, ERK1/2, and SAPK/JNK further potentiates the suppression of TNF-{alpha}, IL-6, and IL-12p40. To accomplish this, splenic DCs were isolated from sham and trauma-hemorrhage mice and stimulated with LPS in the presence of inhibitors of p38 (SB203580), ERK1/2 (PD98059), and SAPK/JNK (SP600125). SB203580 (5 µM), at a dose that did not affect the cell viability, significantly reduced TNF-{alpha}, IL-6, and IL-12p40 production by splenic DCs, irrespective of trauma-hemorrhage (Fig. 5, A, B, and D). DCs from trauma-hemorrhage mice, cultured in the presence of PD98059, showed a further reduction in TNF-{alpha}, IL-6, and IL-12 production (Fig. 5, A, B, and D). Treatment of cells with SAPK/JNK inhibitor SP600125 also reduced TNF-{alpha}, IL-6, and IL-12p40 production (Fig. 5, A, B, and D). We also examined the effect of MAPK inhibitors on IL-10 production by splenic DCs. IL-10 production was significantly suppressed by SB203580, PD98059, or SP600125 in both sham and trauma-hemorrhage groups (Fig. 5C).


Figure 5
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  		Fig. 5. Effects of trauma-hemorrhage and inhibitors of p38 (SB203580; SB), ERK1/2 (PD98059; PD), and SAPK/JNK (SP600125; SP) on LPS-induced TNF-{alpha}, IL-6, IL-10, and IL-12 production by splenic DCs. At 2 h following resuscitation, DCs were purified from splenocytes. Purified CD11c-positive cells were cultured in 96-well tissue culture plates with LPS (10 µg/ml) for 24 h in the presence of specific p38, ERK1/2, or SAPK/JNK inhibitor. The levels of TNF-{alpha} (A), IL-6 (B), and IL-10 (C) in DC supernatant were measured using Cytometric Bead Array (CBA). IL-12p40 (D) levels were measured by ELISA. Data are shown as means ± SE of 6 animals in each group. *P < 0.05 compared with the equivalent sham group; #P < 0.05 compared with LPS group.

 
Effect of trauma-hemorrhage on splenic DC TLR4-MD2 expression. Trauma-hemorrhage produced a significant decrease in splenic DC TLR4-MD-2 surface expression levels when compared with the expression seen in splenic DCs from shams (Fig. 6B). The percentage of TLR4-MD-2-positive cells also decreased significantly following trauma-hemorrhage (Fig. 6C). To determine whether trauma-hemorrhage influenced TLR4 gene expression, we used quantitative RT-PCR to assess changes in expression of TLR4 mRNA in purified splenic DCs. With the use of β2-microglobulin as a housekeeping gene control, a 30% decrease in TLR4 mRNA levels in splenic DCs from trauma-hemorrhage mice was observed compared with shams (Fig. 6D).


Figure 6
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  		Fig. 6. Surface expression of toll-like receptor (TLR)4-MD-2 on splenic DCs following trauma-hemorrhage. At 2 h following resuscitation, DCs were purified from splenocytes by magnetic-activated cell sorting (MACS), stained with antibodies against TLR4-MD-2, and analyzed by flow cytometry. A: representative example of six independent analyses: dotted line, isotype control; black line, sham; gray line, trauma-hemorrhage. Results are presented as MFI (B) and percentage of positive cells (C). D: changes in TLR4 gene expression levels following trauma-hemorrhage. Using quantitative RT-PCR, we measured the TLR4 mRNA at 2 h following resuscitation. β2-microglobulin was used as an internal control. The relative expression levels of individual target genes were normalized to the internal control. Data are shown as means ± SE of 6 animals in each group. *P < 0.05 compared with sham.

 
Effect of trauma-hemorrhage on TLR2 ligand zymosan-induced cytokines production. To determine whether trauma-hemorrhage induces splenic DC hyporesponsiveness to other TLR agonists, we examined the effect of trauma-hemorrhage on TLR2 ligand zymosan-induced cytokine production of splenic DCs. To further ascertain the role of MAPKs of TNF-{alpha}, IL-6, IL-10, and IL-12p40 production in trauma-hemorrhage mice, we examined whether inhibition of p38, ERK1/2, and SAPK/JNK further potentiates the suppression of these cytokines. To accomplish this, splenic DCs were isolated from sham and trauma-hemorrhage mice and stimulated with zymosan in the presence of inhibitors of p38 (SB203580), ERK1/2 (PD98059), and SAPK/JNK (SP600125). SB203580 (5 µM) significantly reduced TNF-{alpha}, IL-6, and IL-12p40 production by splenic DCs, irrespective of trauma-hemorrhage (Fig. 7, A, B, and D). DCs from trauma-hemorrhage mice, cultured in the presence of PD98059, showed a further reduction in TNF-{alpha}, IL-6, and IL-12p40 production (Fig. 7, A, B, and D). Treatment of cells with SAPK/JNK inhibitor SP600125 also reduced TNF-{alpha}, IL-6, and IL-12p40 production (Fig. 7, A, B, and D). The results also showed that IL-10 production by splenic DCs was significantly suppressed by SB203580, PD98059, or SP600125 in both sham and trauma-hemorrhage groups (Fig. 7C).


Figure 7
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  		Fig. 7. Effects of trauma-hemorrhage and inhibitors of p38 (SB203580; SB), ERK1/2 (PD98059; PD), and SAPK/JNK (SP600125; SP) on zymosan-induced TNF-{alpha}, IL-6, IL-10, and IL-12 production by splenic DCs. At 2 h following resuscitation, DCs were purified from splenocytes. Purified CD11c-positive cells were cultured in 96-well tissue culture plates with zymosan (10 µg/ml) for 24 h in the presence of specific p38, ERK1/2, or SAPK/JNK inhibitor. The levels of TNF-{alpha} (A), IL-6 (B), and IL-10 (C) in DC supernatant were measured using CBA. IL-12p40 (D) levels were measured by ELISA. Data are shown as means ± SE of 6 animals in each group. *P < 0.05 compared with the equivalent sham group; #P < 0.05 compared with zymosan group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that trauma-hemorrhage downregulated MAPK activation of splenic DCs. In addition to the decrease of MAPK activation, surface expression of TLR4-MD-2 was also suppressed following trauma-hemorrhage. These changes in MAPK activation and TLR4-MD-2 expression were associated with a suppressed ability of splenic DCs to produce cytokines in response to LPS, a potent TLR4 ligand. As with the TLR4 ligand LPS, TLR2 ligand zymosan also induced cytokine production by splenic DCs. However, trauma-hemorrhage also induced hyporesponsiveness to zymosan. Thus the hyporesponsiveness of splenic DCs, as demonstrated in this study, is likely a factor in the observed immunosuppression seen following trauma-hemorrhage.

The MAPK family includes several subgroups such as JNK, ERK, and p38 MAPK. All share the common property of being activated via three-module phosphorylation cascade, which in turn allows them to phosphorylate a wide range of substrates, including other protein kinases, phospholipases, and transcription factors (14, 24). Although there is some cross-talk and cell-type specificity, the JNK and p38 MAPK cascades are strongly activated by stress stimuli and inflammatory cytokines, whereas the ERK pathway is strongly activated by polypeptide growth factors through receptors for tyrosine kinases (31). It is also well known that the MAPK signaling pathway plays important roles in immune responses (18). The MAPK signaling pathway is a highly conserved pathway that is involved in diverse cellular functions, including cell proliferation, cell differentiation, and apoptosis. Although MAPKs have been implicated in various injury models of innate immune response, little information has been available about how trauma-hemorrhage influences their activation in splenic DCs. Accordingly, we focused on the role of MAPKs in splenic DC TLR4-mediated signaling following trauma-hemorrhage.

Mature DCs highly express both myosine heavy chain and costimulatory molecules, including CD40, CD80, CD83, and CD86, on their surface (7, 9). The maturation process can be initiated by inflammatory stimuli, such as TNF-{alpha}, IL-1β, components of infectious agents such as LPS and unmethylated DNA CpG motif, CD40 ligation, or contact sensitizers (2, 12, 23, 51). Many of these maturation stimuli induce phosphorylation of MAPKs, such as p38 MAPK, ERK, and JNK/SAPK. It is well known that MAPKs have a role in both the maturation and apoptosis of DCs (1, 40, 52). However, MAPKs have been reported to have different roles in DC differentiation. Recent studies have shown a crucial role of p38 MAPK and ERK in the maturation of DCs (20, 40). Since we observed suppressed phosphorylation of p38 MAPK and ERK following trauma-hemorrhage, these pathways may contribute to the impaired maturation of DCs induced by trauma-hemorrhage. In addition, a previous study also demonstrated that the inhibition of ERK, which is activated by various growth factors, decreases the threshold for the induction of apoptosis (40). In contrast, activation of p38 MAPK induces apoptosis of DCs (40). Therefore, the balance between the preference of p38 MAPK and ERK induced by external stress and growth signals, respectively, may determine whether the outcome is toward maturation or apoptosis. We believe that one potential mechanism for the increased splenic DC apoptosis following trauma-hemorrhage is the decreased levels of ERK activation. We further recognize that increased apoptosis may also account for a decrease in DC cytokine production; however, to what extent this versus the other potential mechanisms listed above play a major role remains to be determined.

Trauma-hemorrhage represents a pathological condition associated with a pronounced inflammatory response that is in part mediated by hyperactivity of Kupffer cells (47, 48) and hypoactivity of splenocytes (27) and DCs (28). The DC hypoactivity is associated with the decreased production of the inflammatory mediators, including TNF-{alpha}, IL-6, and IL-12. The MAPKs are important in the regulation of the inflammatory response at the cellular signaling level (13, 21, 36, 44), and the MAPK signal transduction pathway is well established in DCs. Our present results also demonstrated that MAPKs are involved in splenic DC TNF-{alpha}, IL-6, IL-10, and IL-12 production. To determine the mechanism responsible for the trauma-hemorrhage-induced suppression of splenic DC TNF-{alpha}, IL-6, and IL-12 production, we examined the role of p38, ERK1/2, and SAPK/JNK. The results showed that trauma-hemorrhage downregulated p38 and ERK1/2 phosphorylation levels in splenic DCs following activation with LPS. Moreover, LPS-induced TNF-{alpha}, IL-6, and IL-12 synthesis/release was reduced in the presence of p38 or ERK1/2 inhibitors (SB203580 and PD98059, respectively), indicating that LPS-induced cytokine production involves p38 and ERK1/2 activation. These results collectively indicate that p38 and ERK1/2 downregulation play a role in the suppression of TNF-{alpha}, IL-6, and IL-12 production by splenic DCs following trauma-hemorrhage.

The immunoregulatory role of DCs relies mainly on the ligation of specific receptors that initiate and modulate DC maturation. After ligation of the receptors, the different effector DC subsets that promote T-cell responses develop. These DC-priming receptors include pattern recognition receptors, as well as receptors that bind tissue factors that are produced by neighboring cells (25). An important group of pattern recognition receptors are TLR members of the IL-1 receptor (IL-1R)/TLR superfamily. Adaptive immunity to pathogens often starts with the initiation of DC maturation after ligation of TLRs, which therefore are crucial proteins that link innate and adaptive immunity (4). In this study, we treated splenic DCs with the TLR4 ligand LPS. After ligand-mediated dimerization, TLR recruits an adaptor protein MyD88 (10, 26, 39), which then initiates a signaling cascade, including activation of IL-1 receptor-associated kinase 4 (IRAK, 39), TNF-receptor associated factor 6 (TRAF6, 11), and evolutionarily conserved signaling intermediate in toll pathways (ECSIT, 30). Together, they mediate activation of nuclear factor (NF)-{kappa}B and MAPKs (3). Our results indicate that the diminished LPS-induced cytokine production following trauma-hemorrhage was accompanied by downregulation of MAPKs and TLR4-MD-2 expression. The signals through TLR4 activate MAPKs; therefore, there is a possibility that downregulation of the TLR4-MD-2 expression causes suppression of MAPK activation. Furthermore, Frleta et al. (19) reported that a CD40 agonist upregulates TLR4-MD-2 surface expression of murine DCs. However, splenic DCs from trauma-hemorrhage mice express lower CD40 on their surface compared with the corresponding DCs from sham mice (unpublished observations). From these observations, it appears that the downregulated interaction between CD40 and TLR4-MD-2 may also be one of the reasons for LPS hyporesponsiveness following trauma-hemorrhage. Furthermore, hyporesponsiveness of splenic DCs was also found after stimulation with the TLR2 agonist zymosan. Nonetheless, it remains to be determined whether the hyporesponsiveness of splenic DCs following trauma-hemorrhage would also occur after stimulation with other TLR agonists such as TLR5 agonist flagellin and TLR9 agonist CpG.

If one compares the concentrations of IL-10, IL-6, and IL-12p40 produced by DCs with the other studies, it is evident that cytokine productions are quite low. Although the precise reasons why the values are low are not known, one possible reason may be the differences in mouse strain. In this study, we used C3H/HeN mice, whereas most other investigators used C57BL/6 or BALB/c mice; it is well known that there are some differences in cytokine production among mouse strains (32).

With regard to the percentage of cells expressing the TLR4-MD-2, we were surprised to note that a large number of splenic DCs expressed very low levels of the receptor complex sufficient for signal transduction, and this decreased after trauma-hemorrhage. When compared with the percentage of TLR4-MD-2-positive cells, the percentage of p38- and ERK1/2-activated cells was quite large. The reasons for this difference remain unknown; however, there is a possibility that LPS also activates MAPK signal transduction via other receptors in addition to TLR4-MD-2. In fact, a previous study (49) suggested that a structurally heterogeneous complex of four receptors also binds LPS and triggers multiple signaling cascades such as NF-{kappa}B and MAPKs. This heterogeneous receptor complex is composed of heat shock proteins 70 and 90, chemokine receptor 4 (CXCR4), and growth differentiation factor 5 (GDF5). Pfeiffer et al. (42) also described an activation cluster composed of CD14, TLR4, CD55, CD16a, CD11b/CD18, Fc{gamma}-receptors CD32 and CD64, Fc{gamma}RIIIa, CD36 and CD81 after LPS stimulation.

The present study indicates that LPS-induced MAPK activation is impaired and that the major LPS receptor is downregulated. Nonetheless, it could be argued that the first observation is the consequence of the reduced TLR4-MD2 expression and is therefore difficult to interpret. It should be noted, however, that the diminished LPS-induced cytokine production following trauma-hemorrhage was accompanied by downregulation of MAPKs and TLR4-MD-2 expression. However, when compared with the percentage of TLR4-MD-2-positive cells, the percentage of p38- and ERK1/2-activated cells was quite large. Therefore, additional mechanisms may exist for LPS-induced MAPK activation. We believe that downregulation of the TLR4-MD-2 expression is one of the causes of suppressed MAPK activation; however, that per se, is not sufficient to explain the suppressed MAPK activation.

Previous studies have shown that MAPKs mediate the response of T-cells and macrophages to traumatic injury (5, 33, 34). Furthermore, p38 MAPK activation has a critical role in liver macrophage (Kupffer cell) activation following injury (15). However, the role of MAPKs following trauma-hemorrhage has been the subject of conflicting reports. In this regard, trauma-hemorrhage has been shown to prevent or enhance cytokine production and MAPK activation in a cell type-specific manner. For instance, trauma-hemorrhage induces activation of MAPKs in Kupffer cells (47, 48). On the other hand, trauma-hemorrhage suppresses activation of MAPKs in splenocytes (27). Our present results indicate that trauma-hemorrhage induces suppressed MAPK activation in splenic DCs. Numerous observations link trauma-hemorrhage with the opposing effects on proinflammatory cytokine production. For instance, inhibition of splenocytes (27) but augmentation of cytokine release has been reported in Kupffer cells (6). Similarly, trauma-hemorrhage appears to have opposing effects on phosphorylation levels of p38 and ERK1/2. Our study shows inhibition of p38 and ERK1/2 MAPK in splenic DCs following trauma-hemorrhage. These data are in accordance with the results which indicate that trauma-hemorrhage leads to a reduction of p38 and ERK1/2 phosphorylation in splenocytes (27). In contrast, Kupffer cells from trauma-hemorrhage mice displayed an increase in p38 and ERK1/2 phosphorylation levels upon LPS stimulation (47, 48). The similar, opposing effects of trauma-hemorrhage on proinflammatory cytokine production could therefore parallel the similar, opposing effects of trauma-hemorrhage on activation of p38 and ERK1/2.

It could be argued that the present study utilized measurement at a single time point, i.e., at 2 h following resuscitation and thus it remains unknown whether splenic DC functions are altered and MAPK activation and signal transduction persist for a period of time longer than 2 h. Although a time course study of splenic DCs was not carried out, our previous studies using Kupffer cells, splenic, or peritoneal macrophages indicate that the depression in their function that was evident at 2 h was evident for a prolonged period of time and that it took ~7 days for their functions to return to normal (16). Thus, although a time point other than 2 h was not examined in this study, based on our previous studies, it would appear that the deleterious effects of trauma-hemorrhage on splenic DCs would also persist for ~7 days following resuscitation. However, it remains to be determined whether splenic DCs follow the same pattern as Kupffer cells and other macrophages in terms of the duration of altered functions following trauma-hemorrhage.

In conclusion, our findings indicate that trauma-hemorrhage alters the LPS-induced activation of the MAPK cascade that controls different aspects of the LPS hyporesponsiveness of splenic DCs following trauma-hemorrhage. Trauma-hemorrhage appears to induce decreased cross-talk between the MAPK pathways, as well as uncoupling of some LPS response from the MAPK cascade. Hyporesponsiveness of splenic DCs was also found after stimulation with TLR2 agonist zymosan following trauma-hemorrhage. Thus altered MAPK activation and signal transduction contribute to the development of the hyporesponsiveness of splenic DCs that is central in the development of immunological alterations following trauma-hemorrhage.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported by National Institutes of Health Grant RO1 GM-37127.


   ACKNOWLEDGMENTS
 
We thank Dennis Campbell and Bobbi Smith for assistance with manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. H. Chaudry, Center for Surgical Research, Univ. of Alabama at Birmingham, 1670 Univ. Blvd., Volker Hall, Rm. G094, Birmingham, AL 35294-0019 (e-mail: Irshad.Chaudry{at}ccc.uab.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 DISCUSSION
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T. Kawasaki, M. A. Choudhry, M. G. Schwacha, K. I. Bland, and I. H. Chaudry
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